Mechanosensing Controlled Directly by Tyrosine Kinases.

To understand how cells form tissues, we need to understand how the tyrosine kinases are involved in controlling cell mechanics, whether they act directly as parts of mechanosensing machines or indirectly. Cells test the critical parameter of matrix rigidity by locally contracting ("pinching") matrices and measuring forces, and the depletion of contractile units causes transformation. We report here that knocking down the receptor tyrosine kinases (RTKs), AXL, and ROR2, alters rigidity sensing and increases the magnitude or duration of local contraction events, respectively. Phospho-AXL and ROR2 localize to contraction units and bind major contractile components, tropomyosin 2.1 (AXL), myosin IIA (AXL), and filamin A (ROR2). At a molecular level, phosphorylated AXL localizes to active myosin filaments and phosphorylates tropomyosin at a tyrosine critical for adhesion formation. ROR2 binding of ligand is unnecessary, but binding filamin A helps function. Thus, AXL and ROR2 alter rigidity sensing and consequently morphogenic processes by directly controlling local mechanosensory contractions without ligands.

Rab9-dependent retrograde transport and endosomal sorting of the endopeptidase furin.

The endopeptidase furin and the trans-Golgi network protein TGN38 are membrane proteins that recycle between the TGN and plasma membrane. TGN38 is transported by a retromer-dependent pathway from early endosomes to the TGN, whereas the intracellular transport of furin is poorly defined. Here we have identified the itinerary and transport requirements of furin. Using internalisation assays, we show that furin transits the early and late endosomes en route to the TGN. The GTPase Rab9 and the TGN golgin GCC185, components of the late endosome-to-TGN pathway, were required for efficient TGN retrieval of furin. By contrast, TGN38 trafficking was independent of Rab9 and GCC185. To identify the sorting signals for the early endosome-to-TGN pathway, the trafficking of furin-TGN38 chimeras was investigated. The diversion of furin from the Rab9-dependent late-endosome-to-TGN pathway to the retromer-dependent early-endosome-to-TGN pathway required both the transmembrane domain and cytoplasmic tail of TGN38. We present evidence to suggest that the length of the transmembrane domain is a contributing factor in endosomal sorting. Overall, these data show that furin uses the Rab9-dependent pathway from late endosomes and that retrograde transport directly from early endosomes is dependent on both the transmembrane domain and the cytoplasmic tail.

A trans-Golgi network golgin is required for the regulated secretion of TNF in activated macrophages in vivo.

The transmembrane precursor of tumor necrosis factor-alpha (TNF) exits the trans-Golgi network (TGN) in tubular carriers for subsequent trafficking and delivery to the cell surface; however, the molecular machinery responsible for Golgi export is unknown. We previously reported that members of the TGN golgin family are associated with subdomains and tubules of the TGN. Here, we show that the TGN golgin, p230/golgin-245 (p230), is essential for intracellular trafficking and cell surface delivery of TNF in transfected HeLa cells and activated macrophages. Live-cell imaging revealed that TNF transport from the TGN is mediated selectively by tubules and carriers marked by p230. Significantly, LPS activation of macrophages resulted in a dramatic increase of p230-labeled tubules and carriers emerging from the TGN, indicating that macrophages up-regulate the transport pathway for TNF export. Depletion of p230 in LPS-stimulated macrophages reduced cell surface delivery of TNF by >10-fold compared with control cells. To determine whether p230 depletion blocked TNF secretion in vivo, we generated retrogenic mice expressing a microRNA-vector to silence p230. Bone-marrow stem cells were transduced with recombinant retrovirus containing microRNA constructs and transplanted into irradiated recipients. LPS-activated peritoneal macrophages from p230 miRNA retrogenic mice were depleted of p230 and had dramatically reduced levels of cell surface TNF. Overall, these studies have identified p230 as a key regulator of TNF secretion and have shown that LPS activation of macrophages results in increased Golgi carriers for export. Also, we have demonstrated a previously undescribed approach to control cytokine secretion by the specific silencing of trafficking machinery.

Biogenic Synthesis of Silver Nanoparticles with High Antimicrobial and Catalytic Activities using Sheng Di Huang (Rehmannia glutinosa).

Silver nanoparticles (AgNPs) are widely sought after for a variety of biomedical and environmental applications due to their antimicrobial and catalytic properties. We present here a green and simple synthesis of AgNPs utilizing traditional Chinese medicinal herbs. The screening of 20 aqueous herb extracts shows that Sheng Di Huang (Rehmannia glutinosa) had the most promising potential in producing AgNPs of 30±6 nm, with narrow size distribution and high crystallinity. The antimicrobial activities of these AgNPs conducted on E. coli cells were found to be superior in comparison to poly(vinylpyrrolidone)-capped AgNPs synthesized using common chemical method. Additionally, the AgNPs obtained possess excellent catalytic performance in the reduction of 4-nitrophenol to 4-aminophenol. We compared the phytochemical and FTIR spectral analyses of the herb extract before and after synthesis, in order to elucidate the phytochemicals responsible for the reduction of Ag+ ions and the capping of the AgNPs produced.

The golgin GCC88 is required for efficient retrograde transport of cargo from the early endosomes to the trans-Golgi network.

Retrograde transport pathways from early/recycling endosomes to the trans-Golgi network (TGN) are poorly defined. We have investigated the role of TGN golgins in retrograde trafficking. Of the four TGN golgins, p230/golgin-245, golgin-97, GCC185, and GCC88, we show that GCC88 defines a retrograde transport pathway from early endosomes to the TGN. Depletion of GCC88 in HeLa cells by interference RNA resulted in a block in plasma membrane-TGN recycling of two cargo proteins, TGN38 and a CD8 mannose-6-phosphate receptor cytoplasmic tail fusion protein. In GCC88-depleted cells, cargo recycling was blocked in the early endosome. Depletion of GCC88 dramatically altered the TGN localization of the t-SNARE syntaxin 6, a syntaxin required for endosome to TGN transport. Furthermore, the transport block in GCC88-depleted cells was rescued by syntaxin 6 overexpression. Internalized Shiga toxin was efficiently transported from endosomes to the Golgi of GCC88-depleted cells, indicating that Shiga toxin and TGN38 are internalized by distinct retrograde transport pathways. These findings have identified an essential role for GCC88 in the localization of TGN fusion machinery for transport from early endosomes to the TGN, and they have allowed the identification of a retrograde pathway which differentially selects TGN38 and mannose-6-phosphate receptor from Shiga toxin.

Podosome assembly is controlled by the GTPase ARF1 and its nucleotide exchange factor ARNO.

Podosomes represent a class of integrin-mediated cell-matrix adhesions formed by migrating and matrix-degrading cells. We demonstrate that in macrophage-like THP1 cells and fibroblasts stimulated to produce podosomes, down-regulation of the G-protein ARF1 or the ARF1 guanine nucleotide exchange factor, ARNO, by small, interfering RNA or pharmacological inhibitors led to striking podosome elimination. Concomitantly, treatments inducing podosome formation increased the level of guanosine triphosphate (GTP)-bound ARF1. ARNO was found to colocalize with the adhesive rings of podosomes, whereas ARF1 was localized to vesicular structures transiently contacting podosome rings. Inhibition of ARF1 led to an increase in RhoA-GTP levels and triggered assembly of myosin-IIA filaments in THP1 cells, whereas the suppression of myosin-IIA rescued podosome formation regardless of ARF1 inhibition. Finally, expression of constitutively active ARF1 in fibroblasts induced formation of putative podosome precursors: actin-rich puncta coinciding with matrix degradation sites and containing proteins of the podosome core but not of the adhesive ring. Thus, ARNO-ARF1 regulates formation of podosomes by inhibition of RhoA/myosin-II and promotion of actin core assembly.

Mammalian Diaphanous 1 Mediates a Pathway for E-cadherin to Stabilize Epithelial Barriers through Junctional Contractility.

Formins are a diverse class of actin regulators that influence filament dynamics and organization. Several formins have been identified at epithelial adherens junctions, but their functional impact remains incompletely understood. Here, we tested the hypothesis that formins might affect epithelial interactions through junctional contractility. We focused on mDia1, which was recruited to the zonula adherens (ZA) of established Caco-2 monolayers in response to E-cadherin and RhoA. mDia1 was necessary for contractility at the ZA, measured by assays that include a FRET-based sensor that reports molecular-level tension across αE-catenin. This reflected a role in reorganizing F-actin networks to form stable bundles that resisted myosin-induced stress. Finally, we found that the impact of mDia1 ramified beyond adherens junctions to stabilize tight junctions and maintain the epithelial permeability barrier. Therefore, control of tissue barrier function constitutes a pathway for cadherin-based contractility to contribute to the physiology of established epithelia.

Formin DAAM1 Organizes Actin Filaments in the Cytoplasmic Nodal Actin Network.

A nodal cytoplasmic actin network underlies actin cytoplasm cohesion in the absence of stress fibers. We previously described such a network that forms upon Latrunculin A (LatA) treatment, in which formin DAAM1 was localized at these nodes. Knock down of DAAM1 reduced the mobility of actin nodes but the nodes remained. Here we have investigated DAAM1 containing nodes after LatA washout. DAAM1 was found to be distributed between the cytoplasm and the plasma membrane. The membrane binding likely occurs through an interaction with lipid rafts, but is not required for F-actin assembly. Interesting the forced interaction of DAAM1 with plasma membrane through a rapamycin-dependent linkage, enhanced F-actin assembly at the cell membrane (compared to the cytoplasm) after the LatA washout. However, immediately after addition of both rapamycin and LatA, the cytoplasmic actin nodes formed transiently, before DAAM1 moved to the membrane. This was consistent with the idea that DAAM1 was initially anchored to cytoplasmic actin nodes. Further, photoactivatable tracking of DAAM1 showed DAAM1 was immobilized at these actin nodes. Thus, we suggest that DAAM1 organizes actin filaments into a nodal complex, and such nodal complexes seed actin network recovery after actin depolymerization.

Analysis of the local organization and dynamics of cellular actin networks.

Actin filaments, with the aid of multiple accessory proteins, self-assemble into a variety of network patterns. We studied the organization and dynamics of the actin network in nonadhesive regions of cells bridging fibronectin-coated adhesive strips. The network was formed by actin nodes associated with and linked by myosin II and containing the formin disheveled-associated activator of morphogenesis 1 (DAAM1) and the cross-linker filamin A (FlnA). After Latrunculin A (LatA) addition, actin nodes appeared to be more prominent and demonstrated drift-diffusion motion. Superresolution microscopy revealed that, in untreated cells, DAAM1 formed patches with a similar spatial arrangement to the actin nodes. Node movement (diffusion coefficient and velocity) in LatA-treated cells was dependent on the level and activity of myosin IIA, DAAM1, and FlnA. Based on our results, we developed a computational model of the dynamic formin-filamin-actin asters that can self-organize into a contractile actomyosin network. We suggest that such networks are critical for connecting distant parts of the cell to maintain the mechanical coherence of the cytoplasm.

Endosome-to-Golgi transport pathways in physiological processes.

The trans-Golgi network (TGN) is a major traffic hub of the cell, as it regulates membrane transport in the secretory pathway as well as receiving protein cargo by retrograde transport from endocytic compartments. Retrograde transport between endosomes and the TGN is essential for the recycling of membrane proteins which regulate a range of cellular and development functions. In addition, retrograde transport pathways are exploited by many bacterial toxins to mediate cytotoxicity and by some viral proteins to promote pathogenicity. Recent advances using a range of molecular cell biological strategies have identified multiple retrograde transport pathways each regulated by a distinct set of molecular machinery. Here we review recent advances in this field and highlight the importance of these transport pathways in a range of physiological processes.

Identification of different itineraries and retromer components for endosome-to-Golgi transport of TGN38 and Shiga toxin.

The retrograde transport pathways from early/recycling endosomes are critical for recycling a range of endogenous cargo, as well as internalisation of bacterial and plant toxins. We have previously shown that the retrograde transport of the two model cargos, TGN38 and Shiga toxin, differs in the requirement for TGN golgins; transport of TGN38 requires the TGN golgin GCC88 whereas that of Shiga toxin requires GCC185. Here we have further defined the retrograde transport requirements of these two cargos. Tracking the transport of these cargos demonstrated that the bulk of Shiga toxin is transported from early endosomes to recycling endosomes en route to the TGN whereas the bulk of TGN38 is transported from early endosomes to the TGN with only low levels detected in recycling endosomes. In cells depleted of the TGN t-SNARE syntaxin 16, TGN38 accumulated predominantly in early endosomes whereas Shiga toxin accumulated in Rab11-positive recycling endosomes, suggesting distinct routes for each cargo. Retrograde transport of Shiga toxin and TGN38 requires retromer, however, whereas sorting nexin 1 (SNX1) is specifically required for transport of Shiga toxin, sorting nexin 2 (SNX2) is required for the transport of TGN38. Overall, our data have identified different itineraries for the retrograde transport of Shiga toxin and TGN38 and distinct retromer components that regulate the transport of these cargos.

The trans-Golgi network golgin, GCC185, is required for endosome-to-Golgi transport and maintenance of Golgi structure.

Four mammalian golgins are specifically targeted to the trans-Golgi network (TGN) membranes via their C-terminal GRIP domains. The TGN golgins, p230/golgin-245 and golgin-97, are recruited via the GTPase Arl1, whereas the TGN golgin GCC185 is recruited independently of Arl1. Here we show that GCC185 is localized to a region of the TGN distinct from Arl1 and plays an essential role in maintaining the organization of the Golgi apparatus. Using both small interfering RNA (siRNA) and microRNA (miRNA), we show that depletion of GCC185 in HeLa cells frequently resulted in fragmentation of the Golgi apparatus. Golgi apparatus fragments were dispersed throughout the cytoplasm and contained both cis and trans markers. Trafficking of anterograde and retrograde cargo was analysed over an extended period following GCC185 depletion. Early effects of GCC185 depletion included a perturbation in the distribution of the mannose-6-phosphate receptor and a block in shiga toxin trafficking to the Golgi apparatus, which occurred in parallel with the fragmentation of the Golgi ribbon. Internalized shiga toxin accumulated in Rab11-positive endosomes, indicating GCC185 is essential for transport between the recycling endosome and the TGN. In contrast, the plasma membrane-TGN recycling protein TGN38 was efficiently transported into GCC185-depleted Golgi apparatus fragments throughout a 96-h period, and anterograde transport of E-cadherin was functional until a late stage of GCC185 depletion. This study demonstrated (i) a more effective long-term depletion of GCC185 using miRNA than siRNA and (ii) a dual role for the GCC185 golgin in the regulation of endosome-to-TGN membrane transport and in the organization of the Golgi apparatus.